A. Leucovorin Biochemistry: Leucovorin is known to potentiate the activity of 5-fluorouracil (5-FU) in the treatment of colon cancer, elevating 5-FU from a compound of little activity against colon tumors when given alone to one of substantial efficacy when given in combination. However, the order of drug administration is critical--giving 5-FU first results in an ineffective protocol. while biochemical hypotheses have been advanced to explain this effect, they have not been shown to be consistent with the known enzyme chemistry of the folate cycle. Thus leucovorin chemistry is being added to our previously published model of the folate.cycle in human cancer cells. The mode will be used to test the particular hypothesis that leucovorin is converted to other folate forms sufficiently fast to cause an expanded methylene tetrahydrofolate pool capable of enhancing ternary complex formation with tymidylate synthase and 5-FU. B. Folate, Separations: Because it has proved very difficult to separate higher chain-length folate polyglutamates by high performance liquid chromatography, attempts to separate these compounds by supercritical chromatography have been initiated. Folic and dihydrofolic acids and derivatives have been selected for trial separation., Recently, a potential supercritical solvent containing an amine as a solubilizing agent has been identified composed of C02 as the major component together with methanol (a few percent) and methylamine (<l%). The solvent combination is to be examined for its ability to generate sharp folic acid peaks; if successful, the solvent's ability, first, to separate folic acid from dihydrofolate and, second, their polyglutamates from each other will be investigated.